Activation of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (the PTH/PTHrP receptor or PTH1R) by its cognate ligands, PTH or PTHrP, initiates two parallel processes: 1) stimulation of intracellular second messengers leading to biologic effects on mineral ion homeostasis and bone cell development, differentiation, and maturation; and 2)phosphorylation of PTH1R, internalization of the receptor-ligand complexes and desensitization of the biologic responses mediated by this receptor. The physiological role of PTH1R phosphorylation, internalization and desensitization is not known. We have recently mapped the phosphorylation sites on PTH1R and developed a phosphorylation-deficient (pd) PTH1R which is defective in ligand-stimulated internalization. Using homologous recombination techniques we have "knocked in" the mutant pdPTH1R to replace the normal PTH1R. We shall use the pdPTH1R knock in mouse model to understand the role of PTH1R phosphorylation, internalization and desensitization in calcium homeostasis in different physiological condition (Specific Aim 1). We shall also use in vitro cell line models to dissect the molecules involved in PTH1R phosphorylation and internalization (Specific Aim 2). Three naturally-occurring constitutively-active PTH1R mutants provide a unique opportunity to study the molecular mechanism of receptor regulation. The difference in their basal activity and agonist simulation may reflect intrinsic differences in phosphorylation and internalization. Understanding how these mutants are regulated will shed light into the molecular mechanisms of receptor phosphorylation, internalization and desensitization (Specific Aim 3). PTH analogs with unique binding and signaling properties, developed in Project I, will be tested for selective effects on internalization and desensitization. Identification of analogs which differentiate receptor activation from receptor internalization will provide a unique tool to understand the molecular mechanisms of receptor internalization and desensitization (Specific Aim 4).